Biomedical subjects

J J Owen

Publications and source records attributed to J J Owen.

At least 19 recordsLinked to original sources

PubMed1996

Intracellular signaling pathways involved in the induction of apoptosis in immature thymic T lymphocytes.

We describe a novel technique for studying the signaling pathways that control thymocyte negative selection which maintains the essential interactions between thymocytes and thymic stromal cells. Bisected lobes from newborn mouse thymus are maintained in organ culture for up to 36 h, and the thymocytes analyzed by flow cytometry. Inclusion of [3H]inositol during culture allows measurements of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2) hydrolysis and inositol phosphate accumulation. Using this technique we have compared the thymocyte responses induced by anti-CD3, anti-Fas, Con A, and beta-adrenergic stimulation. We show that PtdIns(4,5)P2 hydrolysis precedes anti-CD3-induced thymocyte apoptosis, but not the apoptosis induced by anti-Fas. In contrast, Con A stimulates PtdIns(4,5)P2 hydrolysis, but does not induce thymocyte apoptosis. Anti-CD3, anti-Fas, and Con A all fail to change thymic cAMP levels, but beta-adrenergic stimulation causes a large increase in intracellular cAMP, and agents that elevate cAMP induce thymocyte apoptosis. Inhibition of protein synthesis (with cycloheximide or emetine) prevents the apoptosis induced by anti-CD3 and elevated cAMP, but not that induced by anti-Fas, whereas protease inhibition (with 3,4-dichloroisocoumarin or N(alpha)-tosyl-phenylalanine chloromethyl ketone) prevents the apoptosis caused by all of the effective stimuli. These results offer three important conclusions. First, activation of a variety of different signaling pathways can bring about thymocyte apoptosis. Second, ligation of the thymocyte TCR/CD3 complex provokes PtdIns(4,5)P2 hydrolysis, but signaling through this pathway alone does not necessarily lead to apoptosis. Third, by whichever signaling pathway the response is initiated, the activity of one or more protease enzymes appears to form an essential component in the final common pathway leading to apoptosis.

Adrenergic beta-Agonists↗

PubMed1996

Involvement of LFA-1/ICAM-2 adhesive interactions and PKC in activation-induced cell death following SEB rechallenge.

Ligation of T-cell receptor (TCR) causes mature T cells to proliferate or, on re-exposure to antigen, can cause them to die by activation-induced cell death (AICD). In proliferative responses, costimulatory and adhesive interactions are required and activation of protein kinase C (PKC) has been shown to be essential. Whether or not interactions involving costimulatory signals and PKC have a role in facilitating AICD remains unclear. Here we have examined the role of CD28/B7 and leucocyte function associated antigen-1 (LFA-1)/intracellular adhesion molecule (ICAM) mediated interactions in AICD triggered by staphylococcal enterotoxin B (SEB) in murine lymph node T cells. We show that, after a primary proliferative response to SEB, LFA-1/ICAM-2 adhesive interactions can play a part in AICD following SEB rechallenge, while B7 and ICAM-1 mediated interactions are not essential for this process. In addition, using a highly selective PKC inhibitor, Ro31.8425, we show that PKC activation is essential for the regulation of AICD by SEB rechallenge.

Animals↗

PubMed1996

Cellular interactions in thymocyte development.

Interactions between stromal cells and thymocytes play a crucial role in T cell development. The thymic stroma is complex and consists of epithelial cells derived from the pharyngeal region during development, together with macrophages and dendritic cells of bone marrow origin. In addition, fibroblasts and matrix molecules permeate the whole framework. It is now apparent that these individual stromal components play specialized roles at different stages of T cell differentiation. Thus, at the early CD4-8- stage of development, T cell precursors require fibroblast as well as epithelial cell interactions. Later, at the CD4+8+ stage, as well as providing low avidity TCR/MHC-peptide interactions, thymic epithelial cells have been shown to possess unique properties essential for positive selection. Dendritic cells, on the other hand, are probably efficient mediators of negative selection, but they may not be solely responsible for this activity. Alongside the functional roles of stromal cells, considerable progress is being made in unraveling the nature of the signaling pathways involved in T cell development. Identification of the pre-T cell receptor (pre-TCR) and associated signaling molecules marks an important advance in understanding the mechanisms that control gene rearrangement and allelic exclusion. In addition, a better understanding of the signaling pathways that lead to positive selection on the one hand and negative selection on the other is beginning to emerge. Many issues remain unresolved, and some are discussed in this review. What, for example, is the nature of the chemotactic factor(s) that attract stem cells to the thymus? What is the molecular basis of the essential interactions between early thymocytes and fibroblasts, and early thymocytes and epithelial cells? What is special about cortical epithelial cells in supporting positive selection? These and other issues are ripe for analysis and can now be approached using a combination of modern molecular and cellular techniques.

Animals↗

PubMed1995

Positive selection of thymocytes involves sustained interactions with the thymic microenvironment.

CD4+8+ cortical thymocytes are critically dependent upon interaction with the thymic epithelium to undergo positive selection and maturation into single-positive CD4+ or CD8+ cells. Here we investigate further the nature of this interaction and provide evidence that positive selection requires sustained, rather than "single hit," interaction with thymic stromal cells. We also show that calcineurin-mediated signaling in thymocytes is required for the initial stages of positive selection, but is not essential throughout the period of thymocyte dependence on stromal cell contact during positive selection. In addition, we show that double-positive thymocytes that have initiated positive selection (CD69+4+8+) and newly generated single-positive (CD69+4+) cells differ markedly in response to the same stimulus through the TCR. The former undergo deletion, whereas the latter proliferate, indicating that a critical change in response to TCR ligation occurs within the narrow developmental window between these two stages.

Animals↗

PubMed1995

Stimulation of thymocytes before and after positive selection results in the induction of different NF-kappa B/Rel protein complexes.

Positive selection triggers the differentiation of immature CD4+8+TCRlow thymocytes into TCRhigh single-positive CD4+ or CD8+ cells and is associated with major changes in gene expression. However, little is known about the DNA binding factors controlling these fundamental changes. Here we have examined NF-kappa B/Rel subunit expression and DNA-binding activity in developing thymocytes before and after the induction of positive selection. We show that positive selection is accompanied by the strong up-regulation of c-rel mRNA expression and the constitutive activation of p50/p65 and p50/c-Rel NF-kappa B/Rel complexes, confirming the activation-like status of cells undergoing positive selection. Moreover, CD69+ cells that have initiated positive selection (but not their preselection CD4+8+TCR- precursors) respond to stimulation by the preferential activation of c-Rel-containing DNA-binding complexes. Because the different NF-kappa B/Rel dimers have distinct transcriptional activities and binding site preferences, this preferential activation of c-Rel-containing DNA-binding complexes may well have implications for the changes in gene expression and functional response associated with positive selection.

Amino Acid Sequence↗

PubMed1995

Intracellular signaling events during positive and negative selection of CD4+CD8+ thymocytes in vitro.

We have used in vitro models of thymocyte positive and negative selection in conjunction with selective inhibitors of the TCR-mediated signaling cascade to investigate the intracellular signaling events that mediate these processes. We report that Ro 31.8425, a potent and selective inhibitor of protein kinase C, which blocks the activation of mature T cells in a dose-dependent fashion, has no effect on either positive or negative selection of CD4+8+ thymocytes. In contrast, cyclosporin A fails to prevent negative selection, but inhibits positive selection through a direct effect on developing thymocytes, rather than through the perturbation of stromal cell support. Thus, our data suggest that positive and negative selection may operate via distinct intracellular signaling pathways.

Animals↗

PubMed1995

Phosphatidylinositol 4,5-bisphosphate hydrolysis accompanies T cell receptor-induced apoptosis of murine thymocytes within the thymus.

Regulation of the development of thymocytes into mature T cells within the thymus is now known to involve antigen-induced deletion, by apoptosis, of potentially autoreactive thymocytes, and it can be mimicked either by stimulating the T cell receptor (TcR) complex by monoclonal antibody (mAb) or by ionophore-induced elevation of cytosolic [Ca2+]. To identify signaling pathways employed by the TcR complex of immature thymocytes, we examined the effects of anti-CD3 and anti-TcR beta constant (c) region mAb, staphylococcal enterotoxin B (SEB) and pharmacological agents on the generation of inositol phosphates through hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] both in cultured fetal mouse thymic lobes and in the CD4+CD8+ immature thymocyte cell line, TM10G. Stimulation of the TcR complex with anti-CD3 mAb provoked an accumulation of inositol phosphates diagnostic of the occurrence of receptor-stimulated phosphoinositidase C (PLC) activation. Anti-TcRC beta mAb and SEB provoked smaller but similar responses. The PLC activation evoked by anti-CD3 mAb was suppressed by inhibitors of receptor tyrosine kinases and was unmodified by protein kinase C activation or elevation of cytosolic [Ca2+]. It thus appears that apoptosis triggered by TcR stimulation is associated with PLC activation by a receptor-regulated tyrosine kinase. Treatment of thymic lobes or TM10G cells with fluoroaluminate provoked apoptosis of a wider range of thymocyte subtypes and such stimulation also provoked an accumulation of inositol phosphates. The responses to fluoroaluminate were not prevented by inhibitors of tyrosine kinases, suggesting that unidentified GTP-binding proteins which couple to PLC activation may also be capable of initiating apoptosis by a route independent of the TcR. These results, when considered alongside previous studies of mature T cells, indicate that stimulation of immature thymocytes or of mature T cells through their TcR complex activates the PLC-catalyzed PtdIns(4,5)P2 hydrolysis signaling pathway, and thus that this signaling pathway may be implicated both in provoking apoptosis in immature T cells and in initiating proliferation in mature T cells.

Aluminum Compounds↗

PubMed1995

Thymocyte-stromal-cell interactions and T-cell selection.

Several key advances have been made since 1992, notably in teh area of signalling in early T-cell development and in the regulation of T-cell selection. Future goals include a fuller understanding of the roles of stromal cells and matrix molecules in T-cell development and further elucidation of the diverse signalling pathways involved. The ultimate challenge will be to generate T cells from stem cells under conditions where all of the developmental cues that earmark thymopoiesis are defined.

Animals↗

PubMed1994

Characteristics of an in vitro system of thymocyte positive selection.

We investigated the phenotypic changes accompanying maturation of CD4+CD8+ thymocytes in the presence of thymic stomal cells in vitro. Many of the features that are characteristic of positive selection in vivo, such as an ordered down-regulation of CD4 or CD8 together with up-regulation of the TCR-alpha beta complex, also occurred in vitro. Transient expression of CD69 was also observed, together with activation of IL-2 and IL-4 cytokine genes, providing further evidence that positive selection in vitro involves a pattern of gene regulation comparable to that seen in vivo.

Animals↗

PubMed1994

Thymic epithelial cells provide unique signals for positive selection of CD4+CD8+ thymocytes in vitro.

Using a novel system that supports positive selection in vitro, we have investigated the cellular requirements for this process by testing the ability of individual thymic and nonthymic stromal cell types to support the maturation of CD4+CD8+ thymocytes into CD4+ or CD8+ T cells. We show that thymic cortical epithelial cells are unique in their ability to mediate this maturation, and suggest that in addition to TCR ligation, these cells supply specific signals for positive selection. Moreover, by demonstrating positive selection on ECDI (1-ethyl-3-[3'dimethyl-aminopropyl]-carbodiimide)-fixed epithelial cells in this system, we provide direct evidence that the provision of these signals involves interactions with epithelial cell surface molecules rather than the release of soluble factors.

Animals↗

PubMed1994

Differential expression of Mtv loci in MHC class II-positive thymic stromal cells.

Despite the important role played by endogenous superantigen in shaping the T cell repertoire, little is known concerning the expression of the different Mtv loci in cells of the thymic microenvironment involved in repertoire selection. Here we have examined the expression of a panel of Mtv Ags by different MHC class II+ stromal cel types using reverse transcriptase-PCR and monitored the effects of these stromal cells on the development of cells expressing Mtv-reactive TCR V beta elements in closed thymic organ culture systems. Although Mtv-6 and Mtv-8/9 mRNAs are expressed in normal thymus lobe organ cultures, no Mtv expression was detected in MHC class II+ thymic epithelial cells. In contrast a striking pattern of differential expression was observed in dendritic cells of thymic origin that were devoid of Mtv-8/9 but expressed readily detectable levels of Mtv-6. This pattern of Mtv gene expression correlated well with TCR V beta repertoire development. TCRV beta 3+ T cells, normally deleted in response to Mtv-6, were virtually absent from the single positive thymocyte compartment in thymic organ cultures where dendritic cells are present but were present in reaggregate cultures where the only MHC class II-positive cells were thymic epithelial cells. On the other hand, V beta 11+ T-cells were not deleted in organ cultures, possibly reflecting the absence of Mtv-8/9 expression in dendritic cells. Our studies suggest that the influence Mtvs have on shaping the T cell repertoire not only depends on their expression within a particular strain but also on their tissue specific expression in relation to MHC class II, which is necessary for their presentation.

Animals↗

PubMed1994

Positive selection by purified MHC class II+ thymic epithelial cells in vitro: costimulatory signals mediated by B7 are not involved.

We have investigated the possibility that the costimulatory signals required for activation of mature T cells also play a role in providing differentiation signals for positive selection during T-cell development. We show that purified MHC Class II+ thymic epithelial cells are able to support positive selection in vitro but lack both the functional capacity to deliver costimulatory signals and expression of the costimulatory ligand B7. Our results suggest that the additional signals provided by costimulatory ligands are not required for TCR-mediated positive selection, although other ancillary signals provided by thymic epithelial cells may be involved.

Animals↗

PubMed1994

Developmental regulation of bcl-2 expression in the thymus.

An important factor in shaping the T-cell receptor (TcR) repertoire during thymocyte development is the susceptibility of double-positive (CD4+ CD8+) thymocytes to induction of apoptosis (negative selection) when the TcR is engaged by 'self'-antigens. Recent evidence has suggested that this susceptibility to apoptosis may be influenced by the expression of bcl-2, a proto-oncogene known to increase the resistance to apoptosis in various cell systems. Using a semi-quantitative polymerase chain reaction (PCR) technique in conjunction with staged embryonic material and purified thymocyte subpopulations we have investigated patterns of bcl-2 expression during normal T-cell development. Our results show that while bcl-2 alpha gene expression is readily detectable in immature CD3-CD4-CD8- thymocytes and in mature single-positive TcRhi cells, it is drastically reduced in TcR negative double-positive (CD3- CD4+ CD8+) cortical thymocytes of intermediate maturity. Careful mapping of bcl-2 alpha re-expression in relation to the onset of TcR expression within the population of embryonic thymocytes indicates that bcl-2 alpha is up-regulated as soon as TcR molecules are expressed on the surface of CD4+ CD8+ thymocytes. Therefore, thymocytes susceptible to apoptosis on TcR ligation express bcl-2 alpha mRNA suggesting that changing levels of bcl-2 expression are unlikely to be the only determinant regulating susceptibility to apoptosis in the thymus. The possible implications of these changes in bcl-2 expression regarding other facets of thymocyte development will be discussed.

Animals↗

PubMed1993

MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus.

T lymphocytes are produced in the thymus from precursors originating in the haemopoietic tissues. On entering the thymus, they undergo a programme of proliferation, T-cell receptor (TCR) gene rearrangement, differentiation and repertoire selection. Although the thymus provides a unique environment for these events, the role of the thymic stroma in regulating specific developmental stages is not well understood. We therefore devised an in vitro system to study the role of individual thymic stromal components in T-cell development. We report here that the development of TCR-CD4-CD8-T-cell precursors into TCR+ cells expressing CD4 and/or CD8 requires the presence of both major histocompatibility complex class II+ epithelial cells and fetal mesenchyme. The requirement for mesenchymal support can be mapped to the initial stages of intrathymic development because the later stages of maturation, from double-positive CD4+CD8+ thymocytes into single-positive CD4+ or CD8+ cells, can be supported by epithelial cells alone. We also show that the requirement for mesenchymal cells can be met by cells of the fibroblast line 3T3 (but not by supernatants from these cells). To our knowledge, these findings provide the first direct evidence that mesenchymal as well as epithelial cells are involved in T-cell development, and suggest that their involvement is stage-specific and likely to be dependent on short-range or contact-mediated interactions.

3T3 Cells↗

PubMed1993

Analysis of cytokine gene expression in subpopulations of freshly isolated thymocytes and thymic stromal cells using semiquantitative polymerase chain reaction.

Using a semi-quantitative polymerase chain reaction (PCR) technique we have examined the expression of a panel of cytokines during thymus development, localizing the expression to individual components of the thymic stroma and thymocytes at different maturational stages. The expression of interleukin (IL)-7, stem cell factor (SCF), IL-1 alpha and granulocyte-monocyte-colony-stimulating factor (GM-CSF) mRNA was mapped to individual stromal cell types, while the expression of IL-1 alpha and GM-CSF, along with interferon (IFN)-gamma and IL-4 was detected in the lymphoid compartment of fetal day (Fd) 14 thymus. The expression of lymphoid-specific cytokines genes was selectively down-regulated in thymocytes undergoing maturation. CD3-/lo4+8+ cells, representing an intermediate stage of thymocyte maturation, were devoid of cytokine gene expression. Their CD3+ progeny, on the other hand, expressed IFN-gamma mRNA, supporting the notion that positive selection of cells for further maturation induces the reexpression of some cytokine genes. The cytokine profiles of the various stromal components differed. Purified major histocompatibility complex class II+ cortical epithelial cells strongly expressed IL-7 and SCF, but only limited expression of IL-1 alpha and GM-CSF could be detected. Fetal mesenchyme, on the other hand, expressed SCF, IL-1 alpha and GM-CSF but not IL-7. The importance of these cytokine profiles in relation to T cell development is discussed.

Animals↗

PubMed1992

Apoptosis and T-cell repertoire selection in the thymus.

Thymic tolerance depends on induction of apoptosis (programmed cell death) in immature thymocytes by antigen/MHC complexes on dendritic cells (and possibly other bone marrow-derived APCs). Interactions with antigen/MHC complexes on thymic epithelial cells promote maturation of double-positive thymocytes to single-positive cells. However, the nature of the antigen/MHC complexes on thymic epithelial cells is unknown, and if the stromal cell interaction model as just outlined is correct, then presumably these complexes must be different from those presented on dendritic cells, otherwise all cells signaled for positive selection would be subject to negative selection by similar complexes on APCs. The nature of the signals provided by thymic epithelial cells versus dendritic cells is unknown and may provide a key to understanding the processes of positive and negative selection within the thymus. Clearly the intense selection of the T-cell repertoire within the thymus explains the high level of cell death observed within the immature thymocyte compartment. Such intensive selection shapes the T-cell repertoire in a way that provides an explanation for the genetic basis of immune responsiveness and for the susceptibility of certain individuals to autoimmunity.

Animals↗

PubMed1992

Studies on T cell maturation on defined thymic stromal cell populations in vitro.

We describe an in vitro system in which positive selection of developing T cells takes place on defined stromal cell preparations, which include major histocompatibility complex class II+ epithelial cells but exclude cells of bone marrow origin. In this system, maturation of double-positive T cell receptor negative (TCR-), CD4+8+ thymocytes into single-positive TCR+, CD4+ and CD8+ cells takes place together with the development of functional competence. As in vivo, this maturation is associated with the upregulation of TCR levels as cells progress from double-positive to single-positive status. We also show that class II+ epithelial cells in these cultures are less efficient than dendritic cells in mediating the deletion (negative selection) of V beta 8+ cells by the superantigen staphylococcal enterotoxin B. For the first time, this approach provides a model in which the cellular interactions involved in both positive and negative selection can be studied under controlled in vitro conditions.

Animals↗

PubMed1992

Effects of the thymic microenvironment on the response of thymocytes to stimulation.

We show that, in vitro, the response of thymocytes to certain stimuli, and their survival largely depend on the nature of the culture environment, i.e. whether thymocytes are stimulated within intact thymus lobes or in cell suspension. Exposure of isolated thymocytes to 12-O-tetra-decanoylphorbol 13-acetate (TPA)+ionomycin rapidly abolishes the expression of recombination-activating gene-1 (RAG-1) mRNA (3 h), down-regulates CD4 surface antigen expression (3 h), and enhances apoptosis (24 h). On the other hand, when thymocytes are cultured in intact lobes, TPA plus ionomycin down-regulate rather than abolish RAG-1 mRNA expression (3 h), have little effect on CD4 expression even following 24-h exposure, and only marginally induce apoptosis (24 h). Differences between the culture systems are less pronounced in response to anti-CD3 antibodies. Therefore, it appears that removing thymocytes from their thymic microenvironment makes the cells more susceptible to certain stimuli, possibly by altering their physiological status. In addition, it has been suggested that termination of RAG-1 expression can be linked to thymocyte selection processes. We found that the down-regulation of RAG-1 expression was not dependent on the induction of apoptosis, supporting a proposed link with positive selection.

Animals↗